【作者】 徐艳春；

【导师】 景松岩； 李晓平；

【作者基本信息】 东北林业大学 ， 野生动植物保护与利用， 2001， 博士

【摘要】 本文应用基于荧光标记的全自动DNA序列分析技术和微卫星多态性分析技术，从30个家猫（Felis Catus）微卫星位点中筛选了15个多态性良好、扩增结果稳定可靠的位点用于分析虎的3个亚种，即东北虎（Panthera tigris altaica）、孟加拉虎（P.t．tigris）和华南虎（P.t.amoyensis）的圈养种群的遗传多样性水平。这15位点包括Fca043、Fca074、Fca077、Fca066、Fca090、Foa107、Fca304、F53、F141、F42、Fca391、Fca453、F146、Fca441、Fca41，其中Fca043、Fca074、Fca077、Fca304、Fca453、Fca441、F42等7个位点通过测序分析获得了重复单位的碱基组成和重复次数。对36只虎15个位点的分析发现，这些位点有4-13个等位基因，等位基因频率和基因型频率分布不均衡，10个位点不符和H-W平衡（α=0.01）。对20只东北虎、11只孟加拉虎和5只华南虎的分析表明，群体条合度在物种水平上为0.4937～0.8412之间；在东北虎为0.3691～0.8003之间；在孟加拉虎为0.3872～0.8917之间；在华南虎为0.5341～0.8565之间。杂合子频率与杂合度之间基本吻合，只有少数位点杂合子频率显著低于杂合度。各个亚种都有自己独特的等位基因，通过15个微卫星位点的数据计算亚种间的遗传距离为：东北虎-孟加拉虎之间0.2449、东北虎-华南虎之间0.0687、华南虎-孟加拉虎之间0.1031。两个无关个体15个微卫星位点完全偶合的概率在10^-11～10^-18之间。用15个位点计算的父-子-母三联体的PI值为159824.94～8831296.70，RCP值在0.99999374～0.99999989之间；缺少母本样品时PI为498.23～1448029.34，RCP值为0.99799693～0.99999931之间。不同种属的扩增显示，这些微卫星位点的引物在狍、梅花鹿、家犬、绵羊、人等均不能成功扩增，可以用于野外样品或混合样品的分析。对雄森熊虎山庄的圈养种群的分析表明，圈养种群的管理和遗传多样性保护应从以下4个方面入手：1)严格保持亚种的独立性；2)避免近交繁殖；3)适当延长世代间隔时间；4)促进种群间的基因交流。本文也可为豹等其他大型猫科动物的微卫星遗传标记的研究与应用提供良好的技术路线和基础。更多还原

【Abstract】 By using fluorescent-labeling-based automated DNA sequencing and microsatellite polymorphism analyzing techniques, of 30 microsatellite loci derived from domestic cat (Felts catus), 15 were proved polymorphic, reproducible and reliable in amplifying tiger genomic DNA. They were applied to study the genetic diversity in tiger captive population involving 3 subspecies, Siberian tiger (Pant he ra tigris altaica), Bengal tiger (P t. tigris) and South China tiger (P t. amoyensis). These 15 microsatellites include namely Fca043, Fca074, Fca077, Fca066, FcaO9O, FcalO7, Fca304, F53, F141, F42, Fca391, Fca453, F146, Fca441 and Fca4l, of which 7 loci, Fca043, Fca074, Fca077, Fca304, Fca453, Fca441 and F42 were sequenced to discover the repeating motif and repeating times. In the population analysis of 36 tigers by using 15 microsatellites, 4-13 alleles were found in each locus. The allelic frequency and genotypic frequency of 10 loci showed significant departure from Hardy-Weinberg Balance (a=0.01) indicating the disequilibrium of allelic frequency and genotypic frequency distribution. Heterozygosity was found between 0.4937-4)8412 at species level (im36), 0.3691-41.8003 in Siberian tiger (n20), 0.3872--0.8917 in Bengal tiger (n1 1) and 0.5341桹.8565 in South China tiger (n~5). Observed frequencies of lieterozygotes in each locus basically match the heterozygositv, except for a few loci whose heterozygote frequency is rather lower. Each subspecies o抶ns a few special alleles. Genetic distance among 3 subspecies calculated based on the data of 15 loci is: Siberian tiger-Bengal tiger 0.2449, Siberian tiger-South China tiger 0.0687 and South China tiger-Bengal tiger 0.1031. The matching probability between two unrelated individual at 15 loci is l0l1~~10.i8. P1 value in Father-child-mother set is between ]59824.94~883l296.70, correspondingly, RCP value is between 0.99999374-4)99999989. In the sets without mother抯 sample, P1 is between498.23?448029.34, correspondingly, RCP value is between 0.99799693?.99999931. The study also showed that no successful amplification was detected when applied primer sets of these 15 microsatellites to other species namely roe deer, sika deer, domestic dog, sheep and human DNA. It is suggested that the primers can be reliable in analyzing field samples and mixed or contaminated samples. Taking the example of the captive population of Xiongsen Bear and Tiger Village, this study suggested 4 countermeasures to manage the population and maintain genetic diversity: 1) Keep the independence ot subspecies strictly; 2) avoid inhrecding~ 3) prolong the generation leneth; 4) promote the genetic exchange among populations. As a potential significance, this study can also provide a practical technique route and basis for the microsatellite research in other large felines.更多还原